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| Status |
Public on May 24, 2021 |
| Title |
131283_Axo_LBst50 |
| Sample type |
SRA |
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| Source name |
hindlimb
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| Organism |
Ambystoma mexicanum |
| Characteristics |
tissue: limb bud
Stage: st50
genotype: wt
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single-cell suspensions were obtained by chopping the tissues into <1mm cubes and immediately transferring the cubes to a tube of digesting solution (0.13WU/mL Liberase in 0.7x PBS). The enzymatic digestion was placed on a rotating wheel at room temperature for 45 minutes and then the cells were mechanically dissociated by pipetting 10-15 times with a P1000 pipet tip. Enzymatic reactions were stopped by adding high-serum culture medium (HS-AMEM, 125ml MEM, 20ml heat inactivated A1 FCS, 2ml insulin, 2mL glutamine, 2mL Pen/Strep, 50mL ddH2O), and samples were filtered sequentially through 70 μm and 30 μm MACS® SmartStrainers (Miltenyi Biotec, 130-098-458 and 130-098-462). Dissociated cells were pelleted using 300x rcf centrifugation for 5 minutes at room temperature, then washed twice with 0.7x PBS before resuspension in serum-free medium (114.3ml F12:DMEM+Glutmax, 1.5ml Sodium pyruvate, 1.5ml B27 supplement, 1.5ml MEM NEAA, 1.5ml insulin, 1.5ml Pen/Strep, 28.2mL ddH2O) on ice.
Single-cell cDNA and sequencing libraries were generated following the manufacturer recommendations (10X Genomics, 3’ library kit, v3 chemistry). The quality of the cDNA and resulting sequencing libraries were checked by either bioanalyzer or fragment analyzer.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Cell Ranger (10x Genomics) was used to align sequencing reads originating from Xenopus samples using default parameters. Reads originating from axolotl libraries were mapped using STARsolo (STAR 2.7.5a, Dobin et al., 2013) with parameters mimicking Cell Ranger’s (10x Genomics) transcript counting strategy.
Cellranger and STARsolo generated transcript count matrix files readable by the R package Seurat.
Seurat (v3.1) was used to normalize and scale raw counts which were used for all analyses.
Genome_build: Xenbase v9.2 (Xenopus laevis) (www.xenbase.org, Karimi et al., 2018)
Genome_build: AmexG (Ambystoma mexicanum) (www.axolotl-omics.org, Nowoshilow and Tanaka, 2020)
Supplementary_files_format_and_content: tab-delimited text files include either cellular barcodes or gene features and matrix files contain raw transcript counts for each gene across all cells
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| Submission date |
Feb 01, 2021 |
| Last update date |
May 24, 2021 |
| Contact name |
Tobias Gerber |
| E-mail(s) |
tobias.gerber@embl.de
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| Organization name |
MPI for Evolutionary Anthropology
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| Department |
Genetics
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| Lab |
Barbara Treutlein
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| Street address |
Deutscher Platz 6
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| City |
Leipzig |
| ZIP/Postal code |
04103 |
| Country |
Germany |
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| Platform ID |
GPL27159 |
| Series (1) |
| GSE165901 |
Fibroblast Dedifferentiation as a Determinant of Successful Regeneration |
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| Relations |
| BioSample |
SAMN17726992 |
| SRA |
SRX9994967 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5057662_131283_Axo_LBst50_barcodes.tsv.gz |
17.5 Mb |
(ftp)(http) |
TSV |
| GSM5057662_131283_Axo_LBst50_features.tsv.gz |
483.9 Kb |
(ftp)(http) |
TSV |
| GSM5057662_131283_Axo_LBst50_matrix.mtx.gz |
63.3 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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